Asthmatics are afflicted with acute or chronic infections (e.g., Mycoplasma pneumoniae [Mp], an atypical bacterium) in the airways. To date, the mechanisms by which asthmatics are susceptible to bacterial infections are largely unknown. Airway epithelial cells are critical in host defense against the invading pathogens. Palate, lung, and nasal epithelium clone (PLUNC) proteins are a new family of host defense molecules. Short PLUNC1 (SPLUNC1) is abundantly expressed in large airway epithelium of humans and mice. Preliminary studies from our group demonstrated that: (1) SPLUNC1 protein exists in large quantities in normal, but not allergic, large airway epithelial cells of humans and mice;(2) Human and mouse SPLUNC1 proteins inhibit Mp growth and epithelial IL-8 production upon stimulation with Toll-like receptor 2 (TLR2) ligands;(3) While Mp infection increases epithelial SPLUNC1, allergic inflammation or the Th2 cytokine IL-13 decreases SPLUNC1;and (4) SPLUNC1 induction is dependent on TLR2 signaling activation. These studies led to the following central hypothesis: SPLUNC1 protein is critical in host defense against respiratory bacterial (e.g., Mp) infections. In the presence of a Th2 cytokine milieu, the expected increases of airway epithelial SPLUNC1 production upon an infection are dampened, leading to a persistent airway bacterial infection and inflammation. To test this hypothesis, we propose three aims. Aim 1 is to test whether SPLUNC1 protein exerts host defense and anti-inflammatory functions through: a) direct inhibition of bacterial growth;b) binding to bacterial components (e.g., lipoproteins);and c) binding to host cell receptors (i.e., TLR2) and subsequently inhibiting the signaling of bacteria or bacterial components. We will generate wild-type and mutated SPLUNC1 proteins and soluble TLR2 to test this specific hypothesis. Aim 2 is to test the specific hypothesis that Mp alone increases TLR2 signaling activity, leading to epithelial SPLUNC1 up-regulation. A Th2 cytokine milieu (e.g., IL-13) inhibits Mp-induced TLR2 signaling activation and subsequent SPLUNC1 production, thereby impairing Mp clearance. Transcription factors nuclear factor kappa B (NF-?B) and heat shock factor-1 (HSF-1) are activated upon TLR2 stimulation, and directly enhance SPLUNC1 expression. Primary airway epithelial cell cultures will be performed to tie Mp infection, TLR2 signaling and SPLUNC1 regulation in a Th2 cytokine milieu using molecular methods (e.g., RNA interference). Aim 3 is to use mouse models to support our in vitro data from Aims 1 and 2 in that: 1) SPLUNC1 is critical to lung defense against Mp;2) IL-13 decreases TLR2 signaling, thus impairing lung SPLUNC1 and Mp clearance. TLR2, HSF-1, NF-?B pathway and STAT6 deficient, and IL-13 transgenic mice will be used to prove the role of TLR2 signaling in regulating SPLUNC1 expression and functions under a Th2 cytokine milieu. Our proposed studies will reveal novel molecular mechanisms responsible for an increased susceptibility of asthmatics to infections, which will lead to the discovery of effective therapy in attenuating airway infections. PUBLIC HEALTH RELEVANCE. Despite the use of various commercially available antibiotics, airway bacterial infections continue to pose a significant challenge in lung diseases including asthma. Asthma is a common disease that afflicts people in the US and worldwide. Our research findings will significantly advance our knowledge in understanding the molecular mechanisms responsible for an increased susceptibility of asthmatics to bacterial infections in the lung, thus helping the design of novel therapeutic strategies in attenuating airway infectious and inflammatory processes in asthma and perhaps in other chronic pulmonary diseases.